Aging is the leading risk factor for most of the chronic diseases that account for the bulk of morbidity, mortality, and health costs. It may be feasible to delay age-related diseases as a group by targeting aging mechanisms, such as cellular senescence. We discovered that removing senescent cells enhances healthspan in progeroid INK-ATTAC mice expressing a drug-inducible suicide transgene in senescent cells. Our hypothesis is that pharmacological targeting of survival pathways in senescent cells can be translated into interventions that enhance healthspan. It is crucial to develop compounds that selectively target senescent cells -senolytic agents. Senescent cells are resistant to apoptosis, so we used bioinformatics and RNA interference approaches to identify survival pathways needed for senescent cell survival. We found that prototype senolytic agents targeting these pathways selectively eliminated senescent cells and alleviated a range of aging- and senescence-related phenotypes. Aim 1 is to delineate mechanisms and effects of targeting senescent cells in vitro. We will dissect senescent cell survival mechanisms and senolytic pathways by uncovering new drug targets needed for senescent cell survival by further tracing the pathways through which our current agents exert senolytic effects. We found that genetic and pharmacological targeting of senescent cells both remove ~30-70% -but not all- senescent cells, yet phenotypes are alleviated. Thus, we suspect there are senescent cell subtypes that differ in susceptibility to removal and contribution to adverse phenotypes. We will determine how senescent cells that are eliminated differ from remaining senescent cells in vitro and in vivo. We will leverage our finding that senescent cells arising from different cell types vary in susceptibiity to different senolytic agents to discover cell type-specific senolytic pathways. We will test effecs of combinations of senolytic agents that act through distinct pathways. Aim 2 is to test effectiveness of senolytic agents in clearing senescent cells in vivo. We will compare clearance from different tissues by our senolytic agents in rodents. We found these agents alleviate cardiac dysfunction, impaired vascular reactivity, immobility, frailty, neurologic deficits, osteoporosis, and diabetes in mouse models. We will gauge the strength of associations between senescent cell clearance and phenotype resolution in mice treated with senolytic agents and a novel senescent preadipocyte transplantation model. We will test the extent to which agents exert phenotypic effects by clearing senescent cells vs. effects on non-senescent cells. Aim 3 is to determine the impact of senolytic agents on healthspan phenotypes. Effects of senolytic agents and combinations will be tested on aging metabolic, cardiovascular, lung, muscle, cognitive, and immune phenotypes and in models relevant to chronic diseases. Effects on late life survival in mice will be ascertained. We will test if senolytic agents phenocopy or ar additive (epistasis) to effects of suicide gene-mediated senescent cell clearance. Development of drugs that eliminate senescent cells selectively could lead to a transformation in treating multiple age-related disorders.